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Journal of Bacteriology, December 2005, p. 8462-8469, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8462-8469.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic Requirements for Potassium Ion-Dependent Colony Spreading in Bacillus subtilis

Rebecca F. Kinsinger,¹ Daniel B. Kearns,^2, Marina Hale,¹ and Ray Fall^1*

Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215,¹ Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge, Massachusetts 02138²

Received 26 August 2005/ Accepted 3 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Undomesticated strains of Bacillus subtilis exhibit extensive colony spreading on certain soft agarose media: first the formation of dendritic clusters of cells, followed by spreading (pellicle-like) growth to cover the entire surface. These phases of colonization are dependent on the level of potassium ion (K⁺) but independent of flagella, as verified with a mutant with a hag gene replacement; this latter finding highlights the importance of sliding motility in colony spreading. Exploring the K⁺ requirement, directed mutagenesis of the higher-affinity K⁺ transporter KtrAB, but not the lower-affinity transporter KtrCD, was found to inhibit surface colonization unless sufficient KCl was added. To identify other genes involved in K⁺-dependent colony spreading, transposon insertion mutants in wild-type strain 3610 were screened. Disruption of genes for pyrimidine (pyrB) or purine (purD, purF, purH, purL, purM) biosynthetic pathways abolished the K⁺-dependent spreading phase. Consistent with a requirement for functional nucleic acid biosynthesis, disruption of purine synthesis with the folic acid antagonist sulfamethoxazole also inhibited spreading. Other transposon insertions disrupted acetoin biosynthesis (the alsS gene), acidifying the growth medium, glutamine synthetase (the glnA gene), and two surfactin biosynthetic genes (srfAA, srfAB). This work identified four classes of surface colonization mutants with defective (i) potassium transport, (ii) surfactin formation, (iii) growth rate or yield, or (iv) pH control. Overall, the ability of B. subtilis to colonize surfaces by spreading is highly dependent on balanced nucleotide biosynthesis and nutrient assimilation, which require sufficient K⁺ ions, as well as growth conditions that promote sliding motility.

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is now known that various wild-type Bacillus subtilis strains can form structured multicellular biofilms in the form of floating pellicles on liquid media (3, 4) and pellicle-like surface colonies on semisolid media (18). In the latter case, different phases of colonization can be visualized. In the first phase, cells spread over the surface as dendritic branching colonies. If sufficient potassium ions are present, the cells enter a second growth phase in which the colony can spread over the entire surface as a pellicle-like film. Both phases of surface colonization appear to be independent of flagella and require secretion of surfactin (a lipopeptide with potent surfactant properties [23]). These results are consistent with a sliding motility mechanism, as described by Henrichsen (12), which is defined as surface translocation produced by expansive forces in the growing colony combined with special surface properties to lower the friction between the cells and substrate. However, it is notable that the original observations of sliding motility suggested that a spreading zone of sliding bacteria consists of a single layer of densely packed cells (12). The colony spreading we have observed recently (18) and explore here is not due to expansion of a monolayer of cells but instead represents robust multicellular colony formation akin to that of the pellicles mentioned above.

Although sliding motility is known to occur in a variety of gram-negative and gram-positive bacteria (reviewed by Harshey[10]), surprisingly little is known of the underlying mechanisms of this type of motility. Kolter and coworkers have investigated some aspects of sliding motility in Mycobacterium smegmatis and found that effective colony spreading requires the formation of acetylated glycopeptidolipids in the outermost layer of the cell envelope (24, 25). These workers have proposed that the hydrophobic fatty acyl tails of glycopeptidolipids act to lower the friction to facilitate sliding motility on the surface of the solidified medium. Similarly, in undomesticated B. subtilis strains surface motility is dependent on the secretion of surfactin, mentioned above, but the link between surfactin and surface colonization has focused primarily on flagellum-dependent swarming (14, 17), not sliding motility.

The main goal of the work presented here was to identify genes required for the K⁺-dependent phase of colony spreading. K⁺ ions play an essential role in bacterial physiology, including osmoregulation, maintenance of cellular pH, and signaling, and have specific roles in the structure and function of numerous enzymes (reviewed by Epstein [6]). It is known that the B. subtilis genome encodes a variety of potential K⁺ transporters and channels. Two K⁺ transporters are encoded by the KtrAB (higher-affinity) and KtrCD (lower-affinity) systems and may serve a primary role in osmoregulation in response to hypertonicity (13). Of less certain function in K⁺ transport is a K⁺-stimulated ATPase that is induced by growth at low K⁺ concentration (28). We expected that potassium uptake systems would be required to support sufficient levels of intracellular K⁺ ions for the K⁺-dependent surface colonization, and this was explored by directed mutagenesis. To find other genes required for surface spreading, transposon mutagenesis was conducted in a wild-type B. subtilis strain (16), and mutants were screened for lack of colony spreading in the presence of K⁺ ions. This work marks the beginning of attempts to define the genetic requirements for K⁺-dependent sliding motility and surface colonization by B. subtilis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains and media. B. subtilis strains, listed in Table 1, were routinely grown at 37°C on Luria-Bertani (LB) medium containing (per liter) 10 g tryptone, 5 g yeast extract, and 10 g NaCl, supplemented with 1.5% (wt/vol) agar where needed to make plates. Throughout this work, surface colonization phenotypes were detected by growth on a casein digest-mannitol medium (CM) solidified with 0.3% (wt/vol) agarose (Fisher Scientific; low electroendosmosis grade with a gel strength of >1,000 g cm^–2) as described in detail elsewhere (7); briefly, it contained (per liter) 10 g D-mannitol, 10 g casein digest (N-Z-Amine A; Sigma-Aldrich Co.), and a trace metal mixture (H medium metals) (7). Use of unbuffered CM agarose medium (initial pH 6.6 to 6.8 in different batches of N-Z-Amine A) gave more discrete dendritic growth, while a medium (CMK) with a K⁺-ion source, 7 mM K₂HPO₄ (added separately from a 100x stock; final pH 7.3), produced the most robust surface colonization (7). Here, except for the transposon screening (below), we controlled pH, phosphate, and K⁺-ion levels in the CM-type media by addition of either 7 mM Na₂HPO₄ (CMN medium) or 7 mM K₂HPO₄ (CMK medium). To determine the threshold for K⁺ ion-dependent colony spreading on CMN agarose, various levels of KCl were added to the medium. In other experiments, these media were supplemented with purine or pyrimidine precursors, acetoin, glutamine, or antibiotics from sterile stocks as indicated in the text. Acetoin was added at 20 mM to mimic the maximal level of acetoin released by wild-type B. subtilis strains during assimilation of glucose (29). Soft agarose plates, typically dried 6 to 8 h or overnight in a laminar-flow hood, were inoculated in the center with a sharpened toothpick from regions of uniform growth (overnight, 37°C) on streaked LB agar plates (with antibiotics as necessary); plates dried overnight were usually incubated in a humidified plastic box.

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TABLE 1. B. subtilis strains used in this work

Transposon mutagenesis. For transposon mutagenesis, a temperature-sensitive vector carrying mini-Tn10, pIC333, in wild-type strain 3610 was used; this construct, strain DS1010, and conditions for generating transposon libraries have been described in detail (16). Spectinomycin-resistant colonies, grown by plating on LB agar supplemented with 100 µg ml^–1 spectinomycin followed by overnight growth at 37°C, were replicated with sterile toothpicks onto gridded plates of CM2 (unbuffered) and CMK2 (7 mM K₂HPO₄), which were solidified with twice the level of agarose normally used in the colony-spreading assay (0.6%, wt/vol) to prevent colony overgrowth. After overnight growth at 40°C, the plates were scored and mutants that failed to form a large surface colony (i.e., only showed dendritic growth) were picked and streaked on LB spectinomycin agar. Colonies from these latter plates were inoculated to the center of individual CM and CMK soft agarose (0.3%) plates (in duplicate or triplicate) to confirm the defective K⁺ ion-dependent surface colonization phenotype and to LB agar containing 1 µg ml^–1 erythromycin plus 25 µg ml^–1 lincomycin (macrolide-lincosamide-streptogramin B resistance from pIC333) (31); colonies with macrolide-lincosamide-streptogramin B resistance were discarded. In analysis of a second transposon insertion library, the CM2 and CMK2 plates (0.6% agarose) were supplemented with 1 mM uridine and 1 mM inosine, as precursors of pyrimidines and purines by their respective salvage pathways (33), and 0.1 mM thiamine, which is derived from the purine biosynthetic pathway.

Transposon insertion sites were identified by cloning into Escherichia coli for DNA sequencing as described by Kearns et al. (16). That the transposon and the mutation causing the surface growth defect were inseparable was determined by backcrossing with SPP1-mediated phage transduction for many of the insertions, as described in the text (16).

Construction of other mutant strains. A B. subtilis 168 strain (SF62S) containing a Tn917 insertion in the gene for the TrnA transcription factor was obtained from S. Fisher (Boston University School of Medicine). SF62S, with the genotype tnrA62Tn917erm::Spec trpC2, was used as a source for SPP1-mediated phage transduction of wild-type strain 3610. Potential transductants were screened for spectinomycin resistance (100 µg ml^–1) and sensitivity to erythromycin (5 µg ml^–1). Several isolates were then tested for disruption of the tnrA gene, using inability to grow with a purine (hypoxanthine) as sole N source (27); this generated strain MH11. For the latter growth medium, defined minimal medium H lacking NH₄Cl (8) with 0.2% glucose, 5 mM hypoxanthine, and 1.5% (wt/vol) agar was used, and the plates were supplemented with spectinomycin (100 µg ml^–1). The wild-type strain 3610 grew well on this medium when spectinomycin was deleted.

Strains of 3610 with disruptions in two K⁺ uptake systems, KtrAB and KtrCD (13), were constructed from mutants (in the B. subtilis laboratory strain JH642) obtained from E. Bremer (Philipps University, Marburg, Germany): GHB1, (ktrAB::Neo); GHB6, ktrC::Spec; and GHB12, ktrD::Tet. Each allele was then transferred to the wild-type strain 3610 by SPP1-mediated phage transduction to obtain the following strains: RFKT7, (ktrAB::Neo)1; RFKT22, ktrC::Spec; RFKT31, ktrD::Tet; and RFKT37, ktrC::Spec ktrD::Tet. Strains were grown in media with the appropriate antibiotics: 10 µg ml^–1 neomycin, 10 µg ml^–1 tetracycline, or 100 µg ml^–1 spectinomycin.

Other methods and chemicals. To verify the phenotypes of surfactin mutants, CMN and CMK plates were supplemented with authentic surfactin (Sigma-Aldrich Co.), added to the surface of plates as described previously (18). Surfactin in the cell-free media, prepared by microcentrifugation of the cells, was assayed by a drop-collapse method (2). In this assay, surfactin was used to generate a standard curve, and CMK broth was used to dilute the samples. Flagellar staining utilized the Ryu stain as described by Heimbrook et al. (11). Motile cells from the edges of surface colonies were allowed to move into 10-µl drops of water for approximately 5 min and then each was stained by this procedure. Acetoin, azaserine, sulfamethoxazole, surfactin, and other antibiotics were obtained from Sigma-Aldrich. Monovalent cations, added as the chloride salts, were analytical grade (>99% purity), except for RbCl, which was listed as having 99.8% purity (Alfa Aesar Co.).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Surface colonization phenotypes of potassium transport mutants. Figure 1 (upper panel) illustrates the K⁺ ion-dependent phenotype of the parent wild-type Marburg strain (3610) on semisolid plates of a casein digest-mannitol medium, either unbuffered (CM), buffered with sodium phosphate (CMN), or buffered with potassium phosphate (CMK). As observed previously with another sample of the undomesticated B. subtilis Marburg strain (18), the surface colonies were dendritic in the unbuffered and sodium phosphate-buffered media, but if the medium contained at least 1 mM K⁺ ions, a profuse colony covered the agarose surface after approximately 12 to 14 h. Consistent with previous observations, B. subtilis wild-type cells only rarely exhibit flagella in the spreading phase on CMK media (15), and, using a common flagellar-staining method (10), it was confirmed here that spreading of the 3610 strain is also essentially independent of flagella. In addition, as described in more detail below, profuse colony spreading was also seen in a flagellumless hag mutant constructed in the 3610 strain.

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FIG. 1. Surface colonization of B. subtilis 3610 and a ktrAB mutant on semisolid agarose media is dependent on potassium ions. The casein digest-mannitol agarose media used included CM (low potassium, unbuffered), CMN (CM supplemented with 7 mM Na2HPO4), and CMK (CM supplemented with 7 mM K2HPO4), as described in Materials and Methods. Plates were inoculated in triplicate and incubated 16 h at 37°C. The plates presented are representative of replicate experiments.

As described by Holtmann et al. (13), B. subtilis contains at least two K⁺ uptake systems, a higher-affinity transporter, KtrAB, and a lower-affinity transporter, KtrCD. Both systems are important for rapid response to changing osmotic stress. To test whether either of these potassium uptake systems was required for K⁺-dependent surface spreading, we obtained B. subtilis JH642 mutants with disruptions in either ktrAB, ktrC, or ktrD genes (Table 1) and transduced these markers into the wild-type 3610 genetic background. The ktrAB mutant was deficient in surface growth unless the media were supplemented with 7 to 8 mM K⁺ ions in different experiments (Fig. 1and data not shown). The KCl requirement could not be replaced by LiCl, NaCl, or CsCl, and only trace growth was seen with RbCl (each at 10 mM). These results support the ideas that the ktrAB mutant requires sufficient K⁺ to allow both dendritic growth and colony spreading and that the KtrAB transport system is very specific for K⁺ ions. In comparison, the ktrCD mutant exhibited surface growth phenotypes that were indistinguishable from the parent 3610 strain on CMN and CMK media (data not shown).

Identification of mutants defective in the K⁺ ion-dependent, colony-spreading phenotype. In order to identify additional genes required for K⁺-dependent colony spreading, we conducted transposon mutagenesis with a temperature-sensitive vector carrying mini-Tn10, pIC333, in the 3610 wild-type strain (16). Over 10,000 colonies were screened for the lack of a response to K⁺ ions, and eight mutants were obtained and characterized (Table 2). The growth phenotype of a representative mutant (RFK6) from the screen is dendritic growth both in the absence (CMN plates) and presence (CMK plates) of added K⁺ ions, but incomplete colonization of the surface (Fig. 2). Incomplete colonization was also seen if the plates were incubated for prolonged periods (24 to 72 h, in a humid atmosphere to prevent drying of the plates). Surprisingly, all eight transposon mutations were localized in genes required for biosynthesis of pyrimidines (pyrB) or purines (purD, purF, purH, purL, and purM). In each case, the mutant phenotype on CMK plates could be bypassed by addition of either uracil or uridine (RFK6, a pyrB mutant, as in Fig. 2) or hypoxanthine or inosine (the seven pur mutants, data not shown). It is unlikely that the phenotypes of the transposon insertions were due to polar effects on other unrelated genes, as all genes downstream of the insertions were in operons dedicated to either pyrimidine or purine biosynthesis and could be bypassed by addition of pyrimidine or purine precursors, respectively (33).

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TABLE 2. Summary of B. subtilis colony-spreading mutants isolated from strain 3610

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FIG. 2. Typical surface colonization phenotype of a B. subtilis transposon mutant selected for lack of response to potassium ions. Shown are surface colonies of the pyrB mutant (RFK6; left) or the parent 3610 strain (right) grown on CMN and CMK media with and without uracil (1 mM). CMN (low-potassium) and CMK (high-potassium) agarose media were centrally inoculated and incubated for 16 h at 37°C. The plates presented are representative of replicate experiments; the dendritic growth of the RFK6 mutant on CMN agarose was faint and difficult to photograph, but always visible to the naked eye.

Sulfamethoxazole, a folate antagonist, inhibits colony spreading to reveal underlying dendritic growth in both wild-type and nonflagellated cells. That purine and pyrimidine biosynthesis mutants were defective in colony spreading suggested that interference with nucleic acid biosynthesis might prevent profuse surface colonization. These considerations led us to attempt to create a purine/pyrimidine imbalance with the antifolate drug sulfamethoxazole (SMZ) (20). SMZ inhibits folic acid biosynthesis and interferes with the biosynthesis of purines, pyrimidines, and certain amino acids (21). As shown in Fig. 3, addition of SMZ at sublethal concentrations (10 or 20 µg ml^–1) to CMK agarose medium permitted dendritic growth of strain 3610 but inhibited the profuse surface colonization. At levels of SMZ higher than its MIC in CMK broth (30 µg ml^–1), complete inhibition of both dendritic and colony spreading on CMK agarose occurred (not shown). Essentially, while SMZ reduces growth rate and yield (in broth), it is somewhat unique in that it is much more effective in inhibiting the second phase of growth than other antibiotics like ciprofloxacin, kanamycin, tetracycline, or vancomycin and has much less effect on the first (dendritic) phase than these other antibiotics (R. Kinsinger and R. Fall, unpublished).

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FIG. 3. Effect of the antifolate drug sulfamethoxazole on surface colonization by wild-type B. subtilis 3610 and a flagellumless hag mutant. CMK plates without and with addition of 10 or 20 µg ml–1 SMZ were centrally inoculated and incubated 16 h at 37°C. The plates presented are representative of replicate experiments.

In addition, we wanted to reexamine the possibility that colony spreading in the wild type is due in part to the presence of flagella that are not readily visualized by standard techniques. For this purpose, a flagellumless mutant with a hag gene replacement, constructed in the 3610 genetic background, was examined (17). Spreading motility in the hag mutant was essentially identical to that of the wild type and was similarly inhibited by the addition of SMZ (Fig. 3). Examination of spreading cells at the margins of expanding surface colonies ofthe hag mutant by flagellar staining confirmed the lack of flagella. Therefore, these results confirm the importance of purine biosynthesis on spreading over surfaces by a flagellum-independent mechanism.

Analysis of a second Tn library. In order to identify additional genes required for K⁺ ion-dependent spreading besides pur and pyr, we reasoned that we could eliminate these classes of mutations if pyrimidine (uridine) and purine (inosine) precursors were included in the medium. In addition, since the purine biosynthetic pathway also gives rise to thiamine (33), the screening medium was also supplemented with thiamine. From over 6,000 colonies screened, 18 mutants defective in K⁺ ion-dependent colony spreading were obtained and analyzed (Table 2). Before gene cloning and analysis of transposon insertion sites, all 18 isolates were tested for correction of the colony-spreading defect by addition of surfactin to the CMN and CMK plates. K⁺-dependent spreading motility was rescued by the addition of surfactin for 16 of the isolates, suggesting each contained a disruption of a gene involved in surfactin biosynthesis. Consistent with this prediction, 3 of the 16 surfactin-dependent isolates were determined to contain insertions in two surfactin synthase genes (srfAA and two srfAB mutations; Table 2). As the phenotypes of these insertions could be complemented by addition of extracellular surfactin (see below), we conclude that the srfAA or srfAB mutations were unlikely to have polar effects on unrelated genes.

The two remaining mutants that were not complemented by exogenous surfactin contained transposon insertions in acetolactate synthase (the alsS gene) and glutamine synthetase (glnA) (Table 1), and both transposon insertions were confirmed to be responsible for the nonspreading phenotype by SPP1-mediated backcrosses. As the alsS mutation disrupts formation of the biosynthetic end product acetoin, we attempted to bypass the mutation by the addition of acetoin to the medium. Acetoin addition failed to restore surface colony growth, suggesting that biosynthesis of this compound is not essential for surface-film formation (here and in the rest of the text we refer to surface films as growth that covers the entire surface). Another possible explanation would be that an alsS mutation in B. subtilis also leads to disruption of cytosolic pH control with release of acids to the medium (29). Thus, it was possible that the spreading defect was a result of growth inhibition by low pH. Compared to the wild type, the alsS mutant grew to a lower final cell density and the medium was acidified to pH 5, compared to a final pH of 8 for the wild type. Addition of bis-Tris propane buffer (pH 8.0) was sufficient to restore robust surface spreading to the alsS mutant in the presence of K⁺ ions (see Fig. 5). It is unlikely that an alsS mutation causes significant indirect polar effects, as genes downstream of the insertion are similarly dedicated to acetoin biosynthesis.

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FIG. 5. Surface colonization by B. subtilis mutant RF3K1 (alsS). RF3K1 (alsS) was inoculated in the center of CMN and CMK plates with and without addition of 50 mM bis-Tris propane buffer (pH 8.0), and plates were allowed to grow 18 h at 37°C. For comparison, plates of 3610 from the same experiment (minus bis-Tris propane buffer) are shown; plates of 3610 with this buffer had a similar growth appearance (not shown). The plates presented are representative of replicate experiments.

To explain the phenotype of the glnA mutation, the enzymatic product of the GlnA reaction, glutamine, was added to the medium. In the presence of K⁺ ions, addition of glutamine was sufficient to restore robust surface film formation to the glnA mutant. It is known that expression of glutamine synthetase (GlnA) is necessary for function of the transcription factor TnrA, which is a global regulator of nitrogen metabolism genes (34). When bound to feedback inhibitors, GlnA binds to TnrA and prevents its DNA from binding to genes regulated by a TnrA box sequence (34, 35). To determine if the surface growth effects seen with the glnA mutant were a result of disruption of controls of TnrA, we constructed a tnrA mutant strain (MH11) in the 3610 genetic background. When grown on CMN or CMK plates, the MH11 mutant exhibited slower growth but surface growth phenotypes identical to the parent strain (shown in Fig. 1). These results indicate that lack of spreading in the glnA mutant is dependent on glutamine biosynthesis but independent of TnrA-mediated gene expression. Polar effects can be ruled out in the case of the glnA insertion, as it is the last gene in a two-gene operon (32).

Correlation of mutant phenotypes with surfactin production and growth rates. As the surface growth phenotypes (dendritic and surface film) seen in the transposon mutants might be generated in whole or part by effects on growth rate and/or surfactin secretion, we measured the generation time and surfactin release to the medium for each mutant. The results are summarized in Table 2, and the mutants deficient in K⁺-dependent colony spreading are clustered into two general categories. In the class I mutants, comprised of the srfAA and srfAB mutants, the inability to form dendrites or spread was tightly correlated with a deficiency in surfactin biosynthesis but not growth rate (in CMK broth), compared to the 3610 strain. For this class, the lack of surfactin was sufficient to explain the colony-spreading defect, as exogenous addition of surfactin partially rescued both dendrite growth and spreading (Fig. 4 and data not shown). It should be noted that in experiments like those shown in Fig. 4, we added avery small amount of surfactin to the center of the plate (40µg); if the added surfactin diffuses throughout the plate during growth, its final concentration is >100 times lower than would normally be seen in the wild type (Table 2), which might explain the lack of complete rescue by added surfactin.

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FIG. 4. Surface colonization can be restored to a srfAB mutant by the addition of extracellular surfactin. Twenty microliters of surfactin (2 mg/ml) was spotted in the middle of CMN and CMK plates prior to inoculation (with either RF3S3 [srfAB] or 3610) and incubation for 16 h at 37°C. The plates presented are representative of replicate experiments.

In the class II mutants, the defect in K⁺-dependent spreading was poorly correlated with surfactin production in broth cultures. For example, the pur mutants (with the exception of purM) produced substantial amounts of surfactin, the glnA mutant produced low levels of surfactin, and the pyrB and alsS mutants produced negligible surfactin in liquid medium. In the cases of the purM, pyrB, and alsS mutants, the defect in surfactin biosynthesis was unable to explain the lack of colony spreading, as these mutants lacked the pellicle-like spreading phase even when surfactin was added to the medium. Furthermore, these mutants must produce some surfactin when grown on surfaces, as each displays the dendritic-spreading phenotype that is surfactin dependent (suggested by the results in Fig. 4). Additionally, when 5-µl drops of water were placed near the edges of expanding dendritic colonies of the purM, pyrB, and alsS mutants, the drops immediately collapsed (in contrast to drops placed in uncolonized areas of the agar surface). This drop collapse is due to reduced surface tension by a surfactant and is indicative of some surfactin secretion during dendritic growth on CMK agarose.

The inability of class II mutants to spread over surfaces appeared to be correlated with growth defects (Table 2), and these mutants could be divided into three subcategories. All of the mutants in class IIa exhibited a 3- to 10-fold increase in generation time and a 2- to 13-fold decrease in the final cell yield when grown in CMK broth. Notably, addition of uracil to the pyrB mutant increased the growth rate and yield and rescued K⁺-dependent spreading. Similar enhancements of the growth of several pur mutants in CMK broth were seen by addition of 1 mM hypoxanthine, a purine precursor (data not shown). The alsS mutant belongs in the growth-defect category (class IIb) because while it grows rapidly in broth, the medium becomes acidified, growth is inhibited, and the final cell yield is reduced. The ktrAB mutant also displays a growth defect (class IIc), as it fails to grow unless relatively high concentrations of potassium ions are present. That reduction in growth rate or yield prevents K⁺-dependent surface spreading is consistent with the mechanism of sliding motility, a form of surface translocation driven by the expansive properties of a growing population.

The growth defects of the class II mutants can also account for the variability observed in surfactin biosynthesis in broth. Surfactin production in B. subtilis is controlled by extracellular accumulation of the ComX and competence-stimulating factor peptides in late-growth-phase cultures (5). Attempts to measure surfactin accumulation at a standard cell density proved intractable. Many of the class II mutants grew to much lower cell densities than the parent strain, as the latter only began to accumulate substantial surfactin (>90 µg ml^–1) in CMK broth at cell densities corresponding to optical density values at 600 nm of >4 (>2.6 x 10⁹ cells ml^–1). For this reason, we followed the growth of each strain until it reached stationary phase, and then analyzed surfactin accumulation in the medium. Presumably the final cell density and concentration of ComX/competence-stimulating factor pheromones are insufficient to induce surfactin biosynthesis in broth, but the same amount of pheromones is sufficient to induce surfactin biosynthesis when the volume is restricted by growth on a surface.

Finally, it should be noted that the rates of extension of surface dendrites of class IIa mutants on CMK agarose plates do not correlate with planktonic growth in CMK broth. For example, comparison of three class IIa mutants (with diverse generation times in broth) including those of glnA (70 min), purM (131 min), and pyrB (253 min) revealed that dendritic growth rates from the point of inoculation were approximately the same (12 to 16 h to reach the edges of the petri dishes). These results highlight the abilities of dendritic structures to grow under conditions of severe nutrient deficiency.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, it has become evident that undomesticated B. subtilis strains can form highly structured multicellular communities, both as floating biofilms (pellicles) on liquid media (3, 4) and as spreading surface colonies on semisolid media (7, 18). In each case, two phases of growth are evident, where proliferation of highly ordered long chains or colonies of cells is followed by continued growth of the bundled cells into larger, more robust structures. Fruiting body-like structures manifest when sporulation is localized within the context of the pellicle biofilm. A variety of genes required for B. subtilis pellicles, biofilms, and fruiting body formation have been identified by directed and nondirected mutagenesis, using various phenotypic tests to visualize gene-specific effects on the formation of these complex multicellular structures (reviewed in references 3, 4, 15, and 30). Notably, none of these genes appears to be related to K⁺-ion uptake or homeostasis. Here, we explored the idea that since the two phases of bacterial spreading on agarose media are controlled by the potassium-ion concentration, these conditions could be used to identify additional genes required for pellicle-like colony growth.

As mentioned above, K⁺ ions play numerous essential roles in bacterial physiology, and various potential K⁺ transporters are known to be encoded in the B. subtilis genome (6). In this work, we focused on the two major K⁺ transporters, the KtrAB (higher-affinity) and KtrCD (lower-affinity) systems, that have been characterized in B. subtilis (13). We expected that potassium uptake systems would be required to support sufficient levels of intracellular K⁺ ions for the K⁺-dependent surface colonization. Consistent with our expectations, disruption of the KtrAB, but not KtrCD, transport system prevented growth and colony spreading on the low-potassium casein digest-mannitol agarose media used (Fig. 1). Addition of sufficient levels of KCl (8 to 10 mM to CMN plates) to the ktrAB mutant restored surface growth, likely by use of the lower-affinity KtrCD system. As with the wild-type strain, surface spreading occurs in two phases when high concentrations of potassium are provided to the ktrAB mutant. It is reported that B. subtilis has normal intracellular levels of K⁺ ions of around 350 mM (13), so it is likely that the first phase of growth seen on CM or CMN media in the wild type (with levels of K⁺ ions <1 mM) involves the higher-affinity potassium transport system KtrAB, possibly with limited colony spreading due to the K⁺ limitation. The role, if any, of the KtrCD transport system in these surface growth phenotypes was not evident in these experiments, as a ktrCD mutant was indistinguishable from the parent strain. Perhaps the KtrCD transport system is more important in osmotic stress-induced K⁺ transport (13) than in metabolic processes that support surface translocation. The role of other potential K⁺ uptake systems in this bacterium also needs to be considered (6).

An unbiased transposon mutagenesis screen was conducted to find other genes involved in K⁺-dependent colony spreading. Surprisingly, many mutations that abolished the spreading phase were localized in genes required for biosynthesis of pyrimidines (pyrB) or purines (purD, purF, purH, purL, and purM). The most likely explanation of this result is that enzymes of both pyrimidine and purine biosynthesis are K⁺ ion-dependent, including carbamoyl phosphate synthetase (the first enzyme of pyrimidine biosynthesis) (19) and AICAR (5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside) transformylase-IMP cyclohydrolase, the last enzyme complex of purine biosynthesis (33), which is very likely to require K⁺ ions. This latter conclusion is based on the finding of an essential bound K⁺ ion in the crystal structure of avian AICAR transformylase-IMP cyclohydrolase (9) and the finding that the B. subtilis gene for this enzyme encodes a highly conserved K⁺ ion binding sequence (22). It is notable that all of the pyr and pur mutants obtained here still required K⁺-ion supplementation for complete surface colonization. Colony spreading on CMK medium might be enhanced by the higher levels of K⁺ ions (14 mM) added, which might in turn promote normal cellular levels of K⁺ ions and support balanced nucleotide biosynthesis or other aspects of cellular metabolism. The phenotype of the glnA mutation is likely to be related to an imbalance in pyrimidine and purine biosynthesis, as the glutamine side chain donates N atoms to both pyrimidine and purine rings. The finding that an alsS mutant could also produce a similar surface colonization defect was initially surprising. Acetolactate synthase is involved in postexponential-phase production of acetoin from pyruvate (in liquid media) (26). However, we found that the alsS mutant rapidly acidified the CM and CMK media used here and, thus, it is likely that this defect simply disrupted normal surface growth by such acidification. The alsS gene defect in surface growth was readily overcome by inclusion of a suitable buffer in the medium (Fig. 5).

In toto, the mutant analysis suggests that two factors are important for K⁺-dependent surface spreading: surfactin production and rapid growth. We suggest that the cells require a high concentration of intracellular potassium to support balanced nucleotide biosynthesis for RNA and DNA synthesis and the efficient nutrient assimilation that is necessary for rapid cell division. A high growth yield is also important to attain a sufficient cell density to activate surfactin biosynthesis by quorum signaling molecules. Finally, surfactin is secreted to reduce surface tension and allow spreading, which is driven passively by the expansive forces of colony growth. In this regard, dendritic spreading and K⁺-dependent spreading can be considered two different manifestations of sliding motility that are perhaps differentiated based on two different thresholds of potassium-ion concentration.

It is likely that K⁺-dependent colony spreading is related to the process of pellicle formation (3); as mentioned above, both appear to be two-phase processes initiated by the clustering of cells before a surface colony assembles. Numerous genes necessary for pellicle formation in B. subtilis have been identified (4), and we are currently investigating whether pellicle mutants affect the first or second phase of surface colony spreading in B. subtilis. Finally, as emphasized by Branda et al. (4), surface colonization and pellicle formation by B. subtilis are models for biofilm formation in a gram-positive species. Given recent evidence that wild-type B. subtilis strains are common on plant roots (7) and can form stable biofilms on cultured Arabidopsis roots and also protect such roots from infection by Pseudomonas syringae (1), it seems likely that future work may reveal new aspects of the importance of B. subtilis surface colonization in the rhizosphere and the role(s) of K⁺ ions and K⁺-ion transport in these processes.

 
This work was supported by grants from the National Institutes of Health, including AI055575-01 (to R.F.), GM18568 (to Richard Losick), and National Research Service Award GM66612 (to D.B.K.), as well as a grant from the Department of Energy, Office of Basic Energy Sciences (DE-FG02-03ER15400).

We thank Richard Losick for encouraging this work and Susan Fisher and Erhard Bremer for providing mutant strains.

 
* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215. Phone: (303) 492-7914. Fax: (303) 492-1149. E-mail: fall{at}colorado.edu.

Present address: Department of Biology, Indiana University, Bloomington, Indiana 47405.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, December 2005, p. 8462-8469, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8462-8469.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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